CryI d-endotoxins produced by Bacillus thuringiensis exhibit insecticidal activity against a variety of lepidopteran insect larvae, and the toxin binding sites are major determinants of this activity. CryIA(c) binds to a glycosyl-phosphatidylinositol (GPI)-anchored 120 kDa protein located on the midgut brush border membrane of Manduca sexta. A soluble form of the protein (115 kDa), produced by release from the GPI anchor by phospholipase C treatment, is capable of binding the endotoxin, and a variety of data indicates that the carbohydrate moiety on the protein is involved in the recognition. The aim of this collaborative project is to further characterize the carbohydrate moiety. Initially, the monosaccharide composition of the 115 kDa glycoprotein was determined by high-pH anion exchange chromatography after acid hydrolysis. Proteolytically derived peptides from the 115 kDa protein that are active in binding will also be characterized with respect to carbohydrate composition and sequence. In addition to glycosyl composition analysis by HPAEC, the peptides will also be characterized my MALDI-TOF MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) before and after treatment with peptide-N-glycosidase F, O-glycosidase, and exoglycosidase. The peptide oligosaccharides may be characterized by fluorophore-assisted carbohydrate electrophoresis and sequential exoglycosidase treatment after release from the peptide(s) by N- or O-glycosidase treatment and derivatization with a fluorescent tag.